Material and Methods

Table of Contents

Materials and Methods…………………………………………………………………………………………………… 3

2.1 Composition of the poplar wood pulp for enzymatic hydrolysis………………………………. 3

2.1.1 Determination of ash and dry weight in poplar wood pulp……………………………………. 3

2.1.2 Determination of lignin and sugars in poplar wood pulp……………………………………….. 4

2.2 Enzymatic hydrolysis…………………………………………………………………………………………….. 8

2.2.1    Impact of washing on production of cellulosic sugars…………………………………………. 8

2.2.2    Impact of pH on production of cellulosic sugars………………………………………………… 9

2.2.3    Impact of buffer on production of cellulosic sugars………………………………………….. 10

2.2.4    Impact of enzyme dosage on production of cellulosic sugars…………………………….. 10

2.2.5    Impact of pulp consistency on production of cellulosic sugars…………………………… 11

2.3    Surfactant Improvement………………………………………………………………………………….. 12

2.3.1 Impact of different surfactant (PEG300; PEG 4000; PEG 800; PEG 10.000) on production of cellulosic sugars……………………………………………………………………………………………………………………. 12

2.3.2 Impact of different surfactant different dosages of PEG 4000 on production of cellulosic sugars         13

2.3.3 Impact of different surfactant different type of surfactant on production of cellulosic sugars    14

2.4 The effect of recycling substrate on the production of cellulosic sugars………………….. 18

2.4.1 Bradford assay………………………………………………………………………………………………. 19

2.4.2 Cellulase activity test………………………………………………………………………………………. 20

References…………………………………………………………………………………………………………………… 23

                                                                             

 

 

 

Materials and Methods

2.1 Composition of the poplar wood pulp for enzymatic hydrolysis

The substrate (poplar wood pulp) used in the experiment and the rest of the experiments in this study was obtained from Greenfield. The choice of poplar wood pulp as the substrate for this study was informed by the fact that they are a readily available source of sustainable feedstock in the US (Balatinecz & Kretschmann, 2001; Wang et al., 2012). The experiments on the determination of the composition of poplar wood pulp focused on the ash, lignin and sugar concentration and the dry weight of the substrate. The experiments done to determine the above-stated components of the substrate are described below.

2.1.1 Determination of ash and dry weight in poplar wood pulp

The study determined the amount of ash and dry weight in the substrate based on National Renewable Energy Laboratory guidelines described Sluiter et al., (2008). The guidelines provide standard analytical procedures in the analysis of biomass, which have been widely used in scientific research (Chen et al., 2007; Xu et al., 2010; Chen et al., 2012). In this study, the initial steps of these experiments involved the transferring of crucibles marked using a generic identifier to a muffle furnace at 575 °C for 5 hours after which they were removed and immediately transferred to a desiccator and left until they cooled. The use of generic marker allows the markings to be easily removed at the end of the experiment (Sluiter et al., 2008). The time taken for the crucibles to cool to room temperature was recorded after which the weight of crucibles was determined and the readings recorded. The crucibles were then placed back into the muffle furnace at 575 °C and dried until the weight change was noted to vary by not more than 3 mg upon subsequent heating and weighing cycles after which 2.0 g of the substrate was weighed into the tared crucibles and resulting weight of the crucibles and substrate recorded. All the samples were prepared and analyzed in triplicate. The samples were then ashed using a muffle furnace (575 °C) for 24 hours. The crucible was placed over the flame using an ashing burner and clay triangle with stand after which they were heated until smoke appeared, which was then ignited and allowed to burn until there was no smoke or flame. The crucibles were then allowed to cool after which they were placed in the muffle furnace again for 5 hours after which they were then removed from the furnace and immediately transferred to a desiccator and left until they cooled. The weight of the crucibles containing the ash was then determined and readings recorded and the crucibles with the ash were then placed back into the muffle furnace at 575 °C and dried until the weight change was noted to vary by not more than 3 mg upon subsequent heating cycles. The dry weight (oven dry weight) was then determined based on the following equation (Equation 1):

…….Equation 1

Equation 1 above gives an accurate determination of the dry weight since in takes into consideration the moisture content present in the samples during weighing (Chan et al., 2011).

The total ash in the substrate was determined based on Equation 2 below (Sluiter et al., 2008):

……Equation 2

 

2.1.2 Determination of lignin and sugars in poplar wood pulp

Sample and standard preparation

Lignin is a complex polymer that does not degrade easily (Ghaffar & Fan, 2013). Analysis of lignin therefore requires initial acid hydrolysis steps, which leads to its fractionation to acid soluble and insoluble forms (Zhang et al., 2013). In this study, the samples were prepared by weighing 300 mg of the substrate into a labeled tared pressure tubes and their weight recorded after which 3.00 mL of 72% sulfuric acid was added and mixed using a Teflon stir rod for a period of 60 seconds (Rodrigues et al., 2012). The use of 72% sulfuric acid in the hydrolysis of lignin in this experiment is since the method takes less time and requires lower temperature (Ritter et al., 1932; Sluiter et al., 2008; Tan & Lee, 2014). The pressure tubes with sample and acid mixture were then placed in a water bath set at 30 °C and incubated for 60 minutes as described by Sluiter et al., 2008. During the incubation period, the samples were stirred using Teflon stir rod after every 10 minutes to ensure an even hydrolysis. After the incubation period, the pressure tubes were removed from the water bath and the acid diluted to 4% concentration through the addition of 84.00 mL distilled water using an automatic burette after which the sample was mixed using inversion movements according the procedure described by Tan and Lee (2014). The sugar recovery standards were then prepared by adding 10 mL of distilled water to the weighed sugar after which 348 μL of 72% sulfuric acid was added and resulting standards transferred to a pressure tube and capped tightly. The prepared sugar recovery standards were then stored in the freezer until when required. All the samples in this experiment were prepared in triplicate.

Analysis of lignin content

The analysis of acid insoluble lignin was carried out using gravimetric method (Effland, 1977; Sluiter et al., 2008; Jeong, & Oh, 2011). The prepared samples were autoclaved at 121°C for 1 hour after which they were vacuum filtered through filtering crucibles whose weight had been predetermined and recorded. A filtering flask was used to collect the filtrate from which 50 mL aliquot was stored a sample storage bottle. The solids that remained in the pressure tube were washed into filtering crucible by rinsing the tubes with 50 mL fresh hot distilled water after which the crucible and acid insoluble residue were then dried at 105 °C for 5 hours after which the samples were transferred to the desiccator. Upon cooling, the crucible and dry residue was then weighed and the readings recorded. The crucibles were then placed in a muffle furnace at 575 °C for 24 hours after which they were removed and immediately transferred to the desiccator and left to cool. After cooling to room temperature the weight of each of the crucible and ash was determined and returned to the furnace until the weight change was noted to vary by not more than 3 mg (Sluiter et al., 2008).

The acid soluble lignin in the samples was analyzed using ultraviolet- (UV-) visible spectrophotometric technique described in previous studies (Hyman et al., 2008; Kline et al., 2010). A background of deionized water was conducted on UV-Visible spectrophotometer after which the absorbance of the hydrolysis liquor aliquot that was previous obtained was determined using UV-Visible spectrophotometer at 240 nm. The samples were then diluted using distilled water to obtain an absorbance that ranged between 0.7 to 1.0. All the samples were analyzed in triplicate. The acid soluble lignin present in the substrate was then determined using the Equation 3 below (Sluiter et al., 2008).

….Equation 3

In the equation above UVabs refers to mean average UV-Vis absorbance, dilution refers to the volume of the diluting solvent. ε refers to the absorptivity of biomass at 240 nm (12) while ODWsample and pathlength refer to the weight of the sample and pathlength of the UV-Vis cell as described by Sluiter et al. (2008).

Determination of sugars in the substrate

In this study the sugars present in the substrate was determined using High-performance liquid chromatography (HPLC) technique described by Sluiter et al. (2006) and Foyle et al. (2007). The analysis involved the preparation of calibration standards that contained the sugars that were analyzed as shown in Table 1 below.

Table 1: Sugar Calibration Standards

Sugar component Concentration range (mg/mL)
D-cellobiose 0.1- 4.0
D(+)glucose 0.1- 4.0
D (+)xylose 0.1- 4.0
D (+)galactose 0.1- 4.0
L (+)arabinose 0.1- 4.0
D (+)mannose 0.1- 4.0
Calibration Verification Standards (CVS)

 

2.5

 

A volume of 20 mL of the previously obtained hydrolysis liquor was transferred to a 50 mL Erlenmeyer flask after which the samples were neutralized to pH 7 by adding calcium carbonate slowly while swirling as described by Sluiter et al. (2008). The resulting supernatant was decanted off and passed through a 0.2 μm filter into an autosampler vial for HPLC analysis. All the samples were prepared in triplicate. The standards, CVS, and the substrate samples were analyzed using HPLC (Biorad Aminex HPX-87P column) following the procedure described Nguyen et al. (1998) and Sluiter et al. (2008). The HPLC injection volume was 50 μL while the mobile phase was made up of HPLC grade water. The flow rate was set at 0.6 mL/minute and the column temperature was set between a range of 80 to 85 °C. The HPLC run time was 35 minutes. The amount of sugar in the substrate was then determined based on Equation 3 below (Sluiter et al., 2006).

…..Equation 3

2.2 Enzymatic hydrolysis

The enzymatic hydrolysis experiments were carried out with an aim of determining how the pH, hydrolysis time, enzyme and substrate concentrations, and presence of surfactants influence the efficiency of enzymatic hydrolysis. The five different experiments that were carried out are described below.

2.2.1    Impact of washing on production of cellulosic sugars

A sample of 15 g of poplar pulp was washed using distilled water as described by Lau & Dale (2009) while another 15g of the same substrate was left unwashed. The pH of the washed and unwashed substrate was the adjusted to 5.0 using 2M Sodium hydroxide (Öhgren et al., 2007) after which both substrates were placed into the fridge for overnight. The pH of the substrate was tested after a day in the fridge and readjusted to 5.0 and the original substrate consistency determined using moisture analyzer. A volume of 137.82mL and 201.84mL of acetate buffer was then added to the unwashed and washed substrate respectively to adjust the consistency as indicated by Tu et al. (2007). The addition of the buffer enhanced the desorption of the enzyme as described by Rodrigues et al. (2012). Glass beads (10g) were then added to each substrate in a flask to facilitate mixing. The volume of enzymes added to the flask was then determined based on substrate dry weight using the following equation (Equation 4):

…………Equation 4

The enzyme concentration used in Equation above was 1% and the enzyme % used was expressed in microliters (125 microliters).

The substrate was then pre-warmed in the incubator for 30 minutes after which enzymes (125 microliters of Novozyme, CTec) were added as described by Rodrigues et al. (2015). The substrate was then incubated at 50 °C and a spinning speed of 200 rpm for 96 hours. During the incubation period, samples were periodically taken from the substrate at the fixed time of 24, 48, 72 and 96 hours. The concentration of sugars in the obtained samples was then determined using HPLC technique (1260 Infinity, Agilent Technologies, Santa Clara, CA, USA) that was equipped with a refractive index detector (RID) and an Aminex HPX-87H column (Bio-Rad, Hercules, California, USA) as described by Sluiter et al. (2011) and Eyéghé-Bickong et al. (2012). The mobile phase that was used was made up of 5mM H2SO4 with a flow rate of 0.5 mL/min. The temperature of the column and detector was maintained at 60°C and 35°C, respectively. The HPLC analysis was performed by first adding 1 ml of the sample to well labeled 2 ml centrifuge tubes after which 1 ml of distilled water was added to the tubes. The samples were then centrifuged at 13000g for 3 min after which they were filtrated through a 0.2μm syringe filter before applying to HPLC (Eyéghé-Bickong et al., 2012).

2.2.2    Impact of pH on production of cellulosic sugars

For this experiment, 15g of unwashed poplar pulp with normal pH of 3 and another sample of 15g of unwashed poplar pulp whose pH was adjusted to 5.0 using 2M Sodium hydroxide as described by Öhgren et al., (2007) were used. Both substrates were placed in the fridge for overnight and their pH determined and adjusted after the refrigeration period to initial pH before refrigeration and the original substrate consistency of each substrate determined using moisture analyzer after which 137.82mL acetate buffer was added to each of the substrates (Tu et al., 2007). Ten g of glass beads was then added in a flask to facilitate mixing and the volume of enzymes added to the flask was then determined based on substrate dry weight using Equation 4 above.

The substrate was then pre warmed in the incubator for 30 min after which enzymes were added and incubated as explain under Section 2.2.1 above. The determination of sugars was carried out using the HPLC technique as explain under Section 2.2.1 above.

2.2.3    Impact of buffer on production of cellulosic sugars

The samples used in this experiment included two samples of 15g of unwashed poplar pulp whose pH was adjusted to 5.0 using 2M Sodium hydroxide (Öhgren et al., 2007). Both substrates were placed into the fridge overnight and their pH determined and adjusted after the refrigeration period to initial pH before refrigeration and the original substrate consistency of each substrate determined using moisture analyzer. A volume of 137.82mL of distilled water was then added to one of the 15g of unwashed poplar pulp sample and to the other 15g of unwashed poplar pulp sample, 137.82mL of acetate buffer was added (Tu et al., 2007). Ten g of glass beads was then added in a flask to facilitate mixing and the volume of enzymes added to the flask was then determined based on substrate dry weight using Equation 4 above.

The substrate was pre warmed in an incubator for 30 min after which enzymes were added and incubated as explain under Section 2.2.1 above. The determination of sugars was carried out using the HPLC technique as explain under Section 2.2.1 above.

2.2.4    Impact of enzyme dosage on production of cellulosic sugars

For this experiment, four samples of 15g of unwashed poplar pulp whose pH was adjusted to 5.0 using 2M Sodium hydroxide (Öhgren et al., 2007) were used. All the four samples were placed into the fridge overnight and their pH level determined and adjusted after the refrigeration period to the initial pH before refrigeration and the original substrate consistency of each substrate determined using moisture analyzer. The consistency of the samples was adjusted to 5% by adding 137.82mL of distilled water to all the four samples after which 10 g of glass beads was then added in a flask to facilitate mixing and the volume of enzymes added to the flask was determined based on substrate dry weight using Equation 4 above. However, the enzyme % used in this experiment varied as described below.

The substrate was pre warmed in the incubator for 30 min after which enzymes were added. For this experiment, four different enzyme dosages were used which included the addition of 1% (125microlitter), 5%(625microlitter), 8%(1000microlitter), 10% (1250microlitter) to appropriately labeled sample of 15g unwashed poplar pulp as described by Cannella and Jørgensen (2014). The samples were then incubated as explain under Section 2.2.1 above. The determination of sugars was carried out using the HPLC technique as explain under Section 2.2.1 above.

2.2.5    Impact of pulp consistency on production of cellulosic sugars

The assessment of the impact of pulp consistency on production of cellulosic sugars was carried out according to the procedure described by Zhao et al. (2011).  Three samples of 15g of unwashed poplar pulp whose pH was adjusted to 5.0 using 2M Sodium hydroxide were used. All the three samples were placed into the fridge for overnight and their pH level determined and adjusted after the refrigeration period to initial pH before refrigeration and the original substrate consistency of each substrate determined using moisture analyzer. The consistency of three samples was adjusted as follows: The consistency of 1st sample was adjusted to 5% by adding 137.82mL of distilled water; the consistency of 2nd sample was adjusted to 10% by adding 61.41mL of distilled water while 35.94mL of distilled water was added to the 3rd sample to obtain a consistency of 15% (Tu et al., 2007) after which 10 g of glass beads was added in flask to facilitate mixing and the volume of enzymes added to the flask was determined based on substrate dry weight using Equation 4 above. However, the enzyme % used in this experiment was 5% (625microlitter) for all the samples.

The substrate was pre warmed in an incubator for 30 min after which enzymes (Novozyme (CTec2)) were added and incubated as explain under Section 2.2.1 above. The determination of sugars was carried out using the HPLC technique as explain under Section 2.2.1 above.

2.3       Surfactant Improvement

The surfactant improvement experiments were done with the aim of determining the impact of different types and dosages of surfactants on the production of cellulosic sugars. The surfactants experiments were performed triplicate and each of the experiments are described below.

2.3.1 Impact of different surfactant (PEG300; PEG 4000; PEG 800; PEG 10.000) on production of cellulosic sugars

The impact of surfactants was assessed based on the procedure described by Helle et al., (1993). The pH of six samples of 15g of unwashed poplar pulp was adjusted to 5.0 using 2M Sodium hydroxide (Öhgren et al., 2007) and placed into the fridge for overnight and their pH level determined and adjusted after the refrigeration period to the initial pH before refrigeration and the original substrate consistency of each substrate determined using moisture analyzer. The consistency of the samples was then adjusted to 5% by adding 137.82mL of distilled water to all the six samples after which 10 g of glass beads was then added in a flask to facilitate mixing (Berlin et al., 2006). Different types of surfactants were then added to five of the six samples of 15g unwashed poplar pulp 24 hours prior to the addition of the enzymes (CTec2). To the first sample, 0.15g of PEG 300 was added while a similar amount of PEG4000, PEG8000 and PEG10.000 were added to the 2nd, 3rd, 4th and the 5th sample respectively. The 6th sample was used as the control sample. The six samples were then incubated at 50 °C and at a spinning speed of 200 rpm for 24 hours after which 1 % (125 microliters) of the enzyme was added to the samples. The volume of enzymes added to the flask was determined based on substrate dry weight using Equation 4 above.

The substrates were then pre warmed in an incubator for 30 min after which enzymes were added and incubated as explain under Section 2.2.1 above. The determination of sugars was carried out using the HPLC technique as explain under Section 2.2.1 above.

2.3.2 Impact of different surfactant different dosages of PEG 4000 on production of cellulosic sugars

The impact of surfactant dose on the production of cellulosic sugar was assessed as described by Eriksson et al. (2002) and Börjesson et al. (2007). Four samples of 15g of unwashed poplar pulp whose pH was adjusted to 5.0 using 2M Sodium hydroxide were placed in the fridge for overnight and their pH level determined and adjusted after the refrigeration period to the initial pH before refrigeration and the original substrate consistency of each substrate determined using moisture analyzer. The consistency of the samples was then adjusted to 5% by adding 137.82mL of distilled water to all the four samples after which 10 g of glass beads was then added in a flask to facilitate mixing (Berlin et al., 2006). Different types of surfactants were then added to three of the four 15g of unwashed poplar pulp samples 24 hours prior to the addition of the enzymes. To the first sample, 0.15g of 1% PEG4000 was added, 5% (0.75g) PEG4000 was added to the 2nd sample while 8% (1.2g) PEG4000 was added to the 3rd sample. The 4th sample was used as the control sample. The four samples were then incubated at 50 °C and at a spinning speed of 200 rpm for 24 hours after which 1 % (125 microliters) of the enzyme was added to the samples. The volume of enzymes added to the flask was determined based on substrate dry weight using Equation 4 above.

The substrates were pre warmed in an incubator for 30 min after which enzymes were added and incubated as explain under Section 2.2.1 above. The determination of sugars was carried out using the HPLC technique as explain under Section 2.2.1 above.

2.3.3 Impact of different surfactant different type of surfactant on production of cellulosic sugars

Impact of PEG4000 on production of cellulosic sugars

In this study, four samples of 15g of unwashed poplar pulp whose pH was adjusted to 5.0 using 2M Sodium hydroxide were used to determine the tmpact of PEG4000 on production of cellulosic sugars as described by Börjesson et al. (2007). The samples were placed in the fridge for overnight and their pH level determined and adjusted after the refrigeration period to the initial pH before refrigeration and the original substrate consistency of each substrate determined using moisture analyzer. The consistency of 1st sample was adjusted to 5% by adding 137.82mL of distilled water; the consistency of 2nd sample was adjusted to 10% by adding 61.41mL of distilled water while 35.94mL of distilled water was added to the 3rd sample to obtain a consistency of 15% after which 10 g of glass beads was then added in flask to facilitate mixing. Different types of surfactants were then added to each of the consistencies of the three of the four 15g of unwashed poplar pulp samples 24 hours prior to the addition of the enzymes. To the 5%, 10% and 15% consistencies of the 1st sample, 1% (0.15g) of PEG4000 was added while the 5% (0.75g) PEG4000 was added to the 5%, 10% and 15% consistencies of the 2nd 15g of unwashed poplar pulp sample. An amount of 1.2g of PEG4000 was added to the 5%, 10% and 15% consistencies of the 3rd sample. The 5%, 10% and 15% consistencies of the 4th sample were used as controls. All the samples were prepared in triplicate and incubated at 50 °C and at a spinning speed of 200 rpm for 24 hours after which 1 % (125 microliters) of the enzyme (CTec2) was added to the samples. The volume of enzymes added to the flask was determined based on substrate dry weight using Equation 4 above.

The substrates were pre warmed in an incubator for 30 min after which enzymes were added and incubated as explain under Section 2.2.1 above. The determination of sugars was carried out using the HPLC technique as explain under Section 2.2.1 above.

Impact of PEG8000 on production of cellulosic sugars

For this experiment, four samples of 15g of unwashed poplar pulp whose pH was adjusted to 5.0 using 2M Sodium hydroxide were used. All the four samples were placed in the fridge for overnight and their pH level determined and adjusted after the refrigeration period to initial pH before refrigeration and the original substrate consistency of each substrate determined using moisture analyzer. The consistency of 1st sample was adjusted to 5% by adding 137.82mL of distilled water; the consistency of 2nd sample was adjusted to 10% by adding 61.41mL of distilled water while 35.94mL of distilled water was added to the 3rd sample to obtain a consistency of 15% after which 10 g of glass beads was then added in flask to facilitate mixing. Different types of surfactants were then added to each of the consistencies of the three of the four 15g of unwashed poplar pulp samples 24 hours prior to the addition of the enzymes. To the 5%, 10% and 15% consistencies of the 1st sample, 1% (0.15g) of PEG8000 was added while the 5% (0.75g) PEG8000 was added to the 5%, 10% and 15% consistencies of the 2nd 15g of unwashed poplar pulp sample. An amount of 8g (8%) of PEG8000 was added to the 5%, 10% and 15% consistencies of the 3rd sample. The 5%, 10% and 15% consistencies of the 4th sample were used as controls. All the samples were prepared in triplicate and incubated at 50 °C and at a spinning speed of 200 rpm for 24 hours after which 1 % (125 microliters) of the enzyme was added to the samples. The volume of enzymes added to the flask was determined based on substrate dry weight using Equation 4 above.

The substrates were pre warmed in the incubator for 30 minutes after which enzymes were added and incubated as explain under Section 2.2.1 above. The determination of sugars was carried out using the HPLC technique as explain under Section 2.2.1 above

Impact of Tween 80 on production of cellulosic sugars

The determination of the impact of different amounts of Tween 80 on production of cellulosic sugars was carried out as described by Castanon and Wilke (1981). Four samples of 15g of unwashed poplar pulp whose pH was adjusted to 5.0 using 2M Sodium hydroxide were placed in the fridge for overnight and their pH level determined and adjusted after the refrigeration period to the initial pH before refrigeration and the original substrate consistency of each substrate determined using moisture analyzer. The consistency of 1st sample was adjusted to 5% by adding 137.82mL of distilled water; the consistency of 2nd sample was adjusted to 10% by adding 61.41mL of distilled water while 35.94mL of distilled water was added to the 3rd sample to obtain a consistency of 15% after which 10 g of glass beads was then added in flask to facilitate mixing (Berlin et al., 2006; Tu et al. 2007). Different types of surfactants were then added to each of the consistencies of the three of the four 15g of unwashed poplar pulp samples 24 hours prior to the addition of the enzymes. To the 5%, 10% and 15% consistencies of the 1st sample, 0.1415g of 1% Tween80 was added while 0.7075g of 5% Tween80 was added to the 5%, 10% and 15% consistencies of the 2nd 15g of unwashed poplar pulp sample. An amount of 1.132g of 8% Tween80 was added to the 5%, 10% and 15% consistencies of the 3rd sample. The 5%, 10% and 15% consistencies of the 4th sample were used as controls. All the samples were then incubated at 50 °C and spinning speed of 200 rpm for 24 hours after which 1 % (125 microliters) of the enzyme (CTec2) was added to the samples. The volume of enzymes added to the flask was determined based on substrate dry weight using Equation 4 above.

The substrates were then pre warmed in the incubator for 30 min after which enzymes were added and incubated as explain under Section 2.2.1 above. The determination of sugars was carried out using the HPLC technique as explain under Section 2.2.1 above

Impact of Triton x-100 on production of cellulosic sugars

The pH of four samples of 15g of unwashed poplar pulp adjusted to 5.0 using 2M Sodium hydroxide as described by Öhgren et al. (2007) after which they were placed into the fridge for overnight. The original substrate consistency of each substrate determined using moisture analyzer after which the consistency of 1st sample was adjusted to 5% by adding 137.82mL of distilled water while the consistency of 2nd sample was adjusted to 10% by adding 61.41mL of distilled water. A volume 35.94mL of distilled water was added to the 3rd sample to obtain a consistency of 15% (Tu et al., 2007). Ten g of glass beads was then added in flask to facilitate mixing. The impact of Triton x-100 on production of cellulosic sugars was then assessed as described by Eriksson et al. (2002). Different types of surfactants were then added to each of the consistencies of the three of the four 15g of unwashed poplar pulp samples 24 hours prior to the addition of the enzymes. To the 5%, 10% and 15% consistencies of the 1st sample, 0.1415g of 1% Triton x-100 was added while 0.7075g of 5% Triton x-100 was added to the 5%, 10% and 15% consistencies of the 2nd 15g of unwashed poplar pulp sample. An amount of 1.132g of 8% Triton x-100 was added to the 5%, 10% and 15% consistencies of the 3rd sample. The 5%, 10% and 15% consistencies of the 4th sample were used as controls. All the samples were then incubated at 50 °C and spinning speed of 200 rpm for 24 hours after which 1 % (125 microliters) of the enzyme was added to the samples. The volume of enzymes added to the flask was determined based on substrate dry weight using Equation 4 above.

The substrates were pre warmed in an incubator for 30 min after which enzymes were added and incubated as explain under Section 2.2.1 above. The determination of sugars was carried out using the HPLC technique as explain under Section 2.2.1 above.

2.4 The effect of recycling substrate on the production of cellulosic sugars.

This experiment aimed evaluating the sugar produced by recycled poplar pulp. The experiment was conducted based on procedure used by Kuhad and Singh (2007) and Marques et al., (2008). Two samples of 15g of unwashed poplar pulp whose pH were adjusted to 5.0 using 2M Sodium hydroxide were used. The samples were placed in the fridge overnight and their pH level determined and adjusted after the refrigeration period to the initial pH before refrigeration. The original substrate consistency of the substrates (50.94%) was determined using moisture analyzer after which the consistency was adjusted to 5% for the two samples by adding 137.82mL of distilled water. Then 0.15g of 1% PEG4000 was added to all the substrates except one that was used as the control. All the samples were then incubated at 50 °C and spinning speed to 200 rpm for 24 hours after which 1 % (125 microliters) of the enzyme was added to the samples. The volume of enzymes added to the flask was determined based on substrate dry weight using Equation 4 above. The substrates were pre warmed in an incubator for 30 min after which enzymes were added and incubated as explain under Section 2.2.1 above. The determination of sugars was carried out using the HPLC technique as explain under Section 2.2.1 above.

After 96 hours of hydrolysis, aluminum containers were placed in an oven heated at 105 °C and left overnight after which they were transferred to a desiccator for 30 minute to cool them to room temperature. The filter paper and aluminum containers were then paired and weighed using weighing balance the accuracy of 0.0001g (0.1mg) and the readings recorded. The container was then marked using a pen while a pencil was used to mark the filter. The glass beads were then removed from the slurry of enzymatic hydrolysis and the slurry centrifuged at 40000 rpm for 10 minutes after which all the solids except the glass beads were collected in a container and oven dried (105 °C) overnight (Zheng et al., 2016). The dry mass was then determined following hydrolysis and the liquefied rate calculated. The solid dried pulp was then taken and 2 g weighed. Distilled water (38mL) was then added to the weighed solid dried pulp (2 g) to achieve a 5% consistency after which 0.02 g of fresh PEG400 and 83microlitter of CTec2 enzyme was added and the mixture incubated for 96 hours. After every 24 hours, a sample was drawn from the incubated mixture and analyzed using HPLC (refer to Section 2.2.1). After 96 hours the procedure of mixing solid dried pulp, distilled water, and the enzyme was repeated 2 more times and after each round, the samples drawn after every 24 hours were analyzed using HPLC. The liquid phase obtained from the centrifuging process in the procedure above was collected and serial diluted 10 times. The obtained dilutions were then used in the Bradford assay and Cellulase activity test described below.

2.4.1 Bradford assay

The Bradford assay was carried out by first thawing one tube of BSA standard obtained from a freezer (Montero et al., 1993). The BSA standards were then prepared in a microplate as illustrated in Table 1.

Table 2: The preparation of BSA standards

BSA concentration (mg/mL) Volume of water (uL) Volume of 0.08 mg/mL BSA standard (uL)
0 160 0
0.005 150 10
0.01 140 20
0.015 130 30
0.02 120 40
0.025 110 50
0.03 100 60
0.035 90 70
0.04 80 80

For this experiment, 2 sets of blanks were prepared and the each of standard had a final volume of 160 uL. After the preparation of the standards, 160 uL of each sample solution (dilutions of the liquid phase obtained from the enzymatic hydrolysis as described above) were pipetted into separate microtiter plate wells and 40 uL of dye reagent concentrate added to each well. The samples and reagents were then thoroughly by swirling the contents. The mixture in each of the wells was then incubated at room temperature for at 15 minutes and the absorbance measured at 595 nm.

2.4.2 Cellulase activity test

The activity of the cellulase enzyme (CTec2) was evaluated based on the procedure described by Adney and Baker (1996). The samples (dilutions of the liquid phase obtained from the enzymatic hydrolysis as described above) were transferred into test tubes and 1.0 mL of 0.05 M Na-citrate with pH 4.8 added to the tube. The buffer and the samples were equilibrated to 50 °C. A volume of 0.5 mL of cellulase enzyme (CTec2) diluted in citrate buffer was then added to each of the test tubes and the test tubes incubated at 50 °C for 60 minutes. Immediately after the incubation period, 3.0 mL of DNS reagent was added to each tube and mixed gently to stop the enzyme reaction (Miller, 1959; Dashtban et al., 2010). Two controls and a blank were used in this experiment. The reagent blank was prepared by adding 1.5 mL citrate buffer into an empty test tube. The enzyme control was prepared by adding 1.0 mL citrate buffer and 0.5 mL enzyme dilution into an empty test tube while the substrate control was prepared by adding 1.5 mL citrate buffer and the sample into an empty test tube. The glucose standards, blanks, and controls were treated the same way as the enzyme assay tubes. The glucose standards were also prepared as shown in Table 2 below

Table 3: Glucose standards

Standard Glucose dilutions (glucose/Citrate) (mg/mL) Volume of glucose dilutions (mL) Citrate buffer (mL)
1 6.7 0.5 1
2 5 0.5 1
3 3.3 0.5 1
4 2 0.5 1

 

After stopping the enzyme reaction by the use of 3.0 mL DNS reagent, all the tubes were boiled in hot water for 5.0 minutes after which they were transferred to cold ice-water bath and left to sit for 1 hour after which the content of all the tubes was diluted using water in the ratio of 0.2: 2.5 and mixture mixed by drawing the mixture up and down using pipettor tip. The absorbance reading was then determined. The amount of glucose released was then determined by using a standard curve of the amount of glucose (mg/0.5 mL) versus A540 after deducting the enzyme blank. The enzyme that releases 2.0 mg was determined using a plot of glucose liberated against the logarithm of enzyme concentration after which the cellulase activity was determined using the Equation 5 below (Adney & Baker, 1996):

…………Equation 5

 

 

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